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The effect of biocidal residues on resistance phenotype in escherichia coli

Wesgate, Rebecca 2019. The effect of biocidal residues on resistance phenotype in escherichia coli. MPhil Thesis, Cardiff University.
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Antimicrobial resistance (AMR) poses a threat to worldwide health, in particular in relation to multi-drug resistant organisms. Hygienic cleaning and disinfection can contribute in the prevention of AMR. There is ample evidence to support the use of disinfectants (biocides) in the decrease of healthcare acquired infections (HCAIs) (Weinstein and Hota, 2004, Maillard, 2018, Webber et al., 2015). However, there is also evidence of instances where disinfectant efficacy may be impeded resulting in microbial survival and emerging resistance (A Rutala and J Weber, 2007). Biocides are said to act as a selective pressure that encourages the acquisition of resistance traits in bacterial cells (Qiu et al., 2012). Furthermore, selective pressure may result from the overexposure of very low concentrations of biocides over long periods of time (Andersson et al., 2012, Gullberg et al., 2014, Gullberg et al., 2011, Thomas et al., 2000). Some biocidal products make claims of “residual biocidal activity” whereas efficacy is usually imparted to a much higher concentration. Some microbial populations may survive exposure to low biocide concentrations, and show decreased susceptibility or resistance to a biocide or consequentially other antimicrobials. This study aims to understand differences between bacterial selection and adaptation in Escherichia coli following exposure to realistic residual - during use - chlorhexidine (CHX) or benzalkonium chloride (BZC) concentrations. It was hypothesised that exposure to a high sub-biocide minimum inhibitory concentration (MIC) would exert a selective pressure enabling the least susceptible bacteria to survive resulting in a permanent change of susceptibility phenotype, whereas a low sub-MIC would be conducive to reactive metabolic shifts resulting in a transient change of susceptibility phenotype. iii Baseline biocide (CHX and BZC) and antibiotic susceptibility of E. coli isolates was obtained using a standard micro-dilution broth protocol, and EUCAST protocol. “Residual” CHX concentration left on surface over a 168 hours period was measured by HPLC. The impact of a range of biocide concentrations (including residual CHX ones) on growth kinetics was investigated. Any changes in susceptibility profile was assessed for stability. Efflux activity and metabolic regulation during exposure to low and high sub-CHX MIC were investigated aiming to identify a link with observed changes in susceptibility phenotype. Finally the propensity for different levels of CHX exposure to influence genetic transfer via conjugation was explored. It was demonstrated that a 0.006 ± 0.002 mg/mL is a realistic residual - during use exposure concentration of CHX. This concentration is 99% lower than the concentration initially applied (20 mg/mL). At this residual concentration, it was possible for CHX susceptible bacteria to survive the disinfection process. Five genotypically distinct strains (UCD-CFS ECP-1L3, UCD-CFS ECP-1L4, UCD-CFS ECP-1B2, UCD-CFS ECP-13P5, UCD-CFS ECP-13P4) demonstrated survival after a 5 min but not 24 hours CHX exposure. Surviving bacteria demonstrated elevated MIC and MBC values; the highest fold change was 32-fold (MIC) and 62- fold (MBC). The elevated MIC values obtained were higher than the average concentration of CHX found on surface. Decreases in MIC or MBC values were observed after residual BZC exposure. No stable changes in MIC and MBC were observed after exposure to residual CHX or BZC, but stable changes were observed for antibiotic resistance for amoxicillin/clavulanic acid, ampicillin, cefpodoxime and cephalothin. Efflux activity was observed during exposure to low (0.00005 mg/mL) but not for high (0.002mg/mL) sub-CHX and sub-BZC MIC. It was demonstrated that changes in susceptibility coincided with changes in the ability to metabolise certain substrates including salicin, L-alanine, betain, creatanine and iv phenylethlalamine. These substances were linked to cell wall and stress signalling regulatory processes. It was surmised that E. coli was able to adapt through metabolic alterations to produce transient changes in CHX susceptibility and stable changes in antibiotic susceptibility. Furthermore, our results show that a transiently adapted population may be selected amongst less tolerant sub-populations at the established CHX-during use concentration. Overall, this work suggests that the intended application concentration of a biocide may in fact be lower than the MIC of target organisms. It is concluded that residual concentrations of biocides do have the potential to drive resistance, particularly stable cross-resistance to antibiotics, through prolonged exposure to low level during use concentrations, driving metabolic modifications of the cell envelope. The potential risk of cross-resistance warrants further investigation.

Item Type: Thesis (MPhil)
Date Type: Completion
Status: Unpublished
Schools: Pharmacy
Subjects: Q Science > Q Science (General)
Date of First Compliant Deposit: 5 December 2019
Last Modified: 05 Dec 2019 10:36

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